5.4.7
POSITIONAL CLONING, MOLECULAR CHARACTERIZATION AND HETEROLOGOUS EXPRESSION OF THE TOSPOVIRUS RESISTANCE GENE SW-5

SH BROMMONSCHENKEL1, SD TANKSLEY2, ANNE FRARY2, AMY FRARY2, WC OTONI3 and A CHEAVEGATTI1

1Department of Plant Pathology/BIOAGRO, Federal University of Višosa, 36571-000 Višosa, MG, Brazil; 2Department of Plant Breeding, Cornell University, 14853 Ithaca, NY, USA; 3Department of Plant Biology/BIOAGRO, Federal University of Višosa, 36571-000 Višosa, MG, Brazil

Background and objectives
The gene Sw-5 introgressed into cultivated tomato (Lycopersicon esculentum) from the wild tomato species L. peruvianum provides resistance against tomato spotted wilt tospovirus (TSWV), tomato chlorotic spot tospovirus (TCSV), groundnut ringspot tospovirus (GRSV) and Chrysanthemum stem necrosis virus (CSNV) [1]. The resistant phenotype conferred by Sw-5 ranges from a hypersensitive-like response to apparent immunity. In order to understand the molecular basis of this broad-spectrum tospovirus resistance, we have isolated and characterized Sw-5 using a positional cloning strategy. In addition, we have tested the effect of Sw-5 in tospovirus-susceptible tobacco plants.

Results and conclusions
Sw-5 was initially mapped to the sub-telomeric region of the long arm of tomato chromosome 9, very tightly linked to the most distal RFLP marker CT220 (0.15 cM, p<0.05) [1]. In the next step of the strategy, two yeast artificial chromosomes (YACs) clones, TY235 (250 kb) and TY257 (400 kb), containing sequences homologous to CT220, were isolated. RFLP mapping of the YAC ends and internal probes demonstrated that YAC 257 spans the Sw-5 locus. The genomic location of Sw-5 was then further refined by assembling a cosmid contig spanning the recombination events proximal to this locus. After analysing more than 5000 F2 plants for recombination events close to Sw-5, the position of the locus was genetically delimited to a maximal chromosomal segment of 100 kb covered by nine overlapping binary cosmid clones. These cosmid clones were then used in Agrobacterium-mediated transformation experiments to identify clones that could complement the tospovirus-susceptible phenotype of the tomato cultivar Moneymaker. These experiments have localized the Sw-5 locus to a 16-kb cosmid. This region was determined to encode two different transcripts, TCD1 and TCD11, based on screening of a leaf cDNA library. Additional transformation experiments showed that the TCD1 corresponds to Sw-5, providing resistance to TSWV, TCSV, GRSV and CSNV in transgenic Moneymaker plants. The deduced sequence of Sw-5 encodes a protein with nucleotide-binding site and leucine-rich domains with similarity to PRF, I2C and RPM1 proteins. Sequence comparison of Sw-5 and sw-5 alleles suggests that the LRR domain could be involved in the resistance specificity of Sw-5. Heterologous expression and functional analysis have shown that Sw-5 provides a limited spectrum of tospovirus resistance in transgenic Xanthi NN tobacco plants. The tobacco plants transformed with the Sw-5 genomic clone displayed a rapid HR only when inoculated with TSWV.

References
1. Brommonschenkel SH, Tanksley SD, 1997. Molecular and General Genetics 256, 121-126 .